Elevated cell surface levels of the human epidermal growth factor receptor subunit 3 (HER3) is associated with resistance to a number of signal-blocking breast cancer treatments, including inhibitors of EGF-R (lapatinib), HER2 (lapatinib, trastuzumab, T-DM1), HER2-3 (pertuzumab), and combination therapy. Additionally, HER3 elevation has been identified on untarget-able tumors such as triple-negative breast cancer (TNBC), including TNBC with acquired resistance to EGF-R inhibition. Patients with such refractory tumors currently have limited treatment options and a poor prognosis. Moreover, as up to 70% of cases resist or acquire resistance to signal-blocking therapies, an alternative approach addressing this important clinical problem has the potential for significant clinical impact. We have developed a self-assembling nanobiological particle, HerDox, which uses HER3 as a portal for targeted entry of toxic molecules. In contrast to receptor-targeted antibodies and tyrosine kinase inhibitors currently used in the clinic, HerDox circumvents the need to modulate signaling and can induce rapid entry of toxic molecules into tumor cells by receptor-mediated endocytosis and membrane penetration. We have previously shown that HerDox can elicit targeted toxicity to HER2+ tumors due to the prevalence of HER2-3 heterodimers on the tumor cell surface, while sparing heart and liver tissue. We are currently exploring the capacity for HerDox to target toxicity to tumors that resist HER2 inhibitors. Our preliminary studies show that HerDox exhibits improved therapeutic efficacy on tumor cell lines with acquired resistance to HER2 inhibitors over parental tumor cells, due in part to the elevated HER3 expression associated with the HER2+ drug resistance. Here we will evaluate the mechanism and efficacy of HerDox in several in vitro and in vivo models representing distinct scenarios along the spectrum of resistance. We will probe the additional factors contributing to the high efficacy of HerDox on tumors with acquired resistance, as these cells exhibit moderately elevated levels of HER3. We will evaluate the efficacy of using signal-inhibitors as adjuvants for HerDox treatment on both nave and inherently-resistant tumors, given the preliminary finding that pretreatment of parental breast cancer cell lines in vitro with trastuzuma induced elevation of HER3. We will also evaluate the efficacy of HerDox on TNBC in an immunocompetent model of disseminated breast cancer and assess whether EGF-R inhibition further sensitizes these tumors to HerDox via HER3 elevation. In all aims of this study, HerDox will be evaluated in comparison to trastuzumab, lapatinib, pertuzumab, T-DM1, and combination treatments. The advantages over signal-blocking inhibitors seen in our preliminary studies suggest that HerDox is likely to present an effective approach in addressing tumors that resist current targeted treatments used in the clinic.